Proteinase inhibitors play a critical role in the regulation of several physiological processes such as blood coagulation, complement fixation, fibrinolysis, and fertilization (Bode and Huber, Biochim. Biophys. Acta 1477:241-252, 2000). Most of these inhibitors are proteins having characteristic polypeptide scaffolds, and are grouped into a number of families including the Kunitz (Laskowski and Kato, Ann. Rev. Biochem. 49: 593-626, 1980), Kazal (Laskowski and Kato, Ann. Rev. Biochem. 49: 593-626, 1980), Serpin (Potempa et al., J. Biol. Chem. 269: 15957-159, 60, 1994) and mucus (Wiedow et al., J. Biol. Chem. 265: 14791-14795, 1990) families.
The Kunitz-type family comprises serine proteinase inhibitors that include one or more Kunitz-type inhibitory domains. Bovine pancreatic trypsin inhibitor (BPTI) is the prototypical Kunitz-type inhibitor. The Kunitz-type family also includes tissue factor pathway inhibitor (TFPI) and type-2 tissue factor pathway inhibitor (TFPI-2). These two inhibitors have been investigated extensively in the past decade, and have been shown to play an important role in inhibiting serine proteinases involved in coagulation and fibrinolysis (Girard et al., Nature 338:518-520, 1989; Broze et al., Biochemistry 29: 7539-7546, 1990; Sprecher et al., Proc. Natl. Acad. Sci. USA 91: 3353-3357, 1994; Bajaj et al., Thromb. Haemost. 86:959-972, 2001).
Human TFPI-2, originally isolated from placenta and designated as placental protein 5 (PP5), is a matrix-associated inhibitor consisting of three tandemly arranged Kunitz-type proteinase inhibitor domains flanked by a short acidic amino terminus and a highly basic carboxy-terminal tail (Sprecher et al., Proc. Natl. Acad. Sci. USA 91:3353-3357, 1994; Miyagi et al., J. Biochem. 116:939-942, 1994) (see FIG. 1). A wide variety of cells including keratinocytes (Rao et al., J. Invest. Dermatol. 104: 379-383, 1995), dermal fibroblasts (Rao et al. J. Invest. Dermatol. 104: 379-383, 1995), smooth muscle cells (Herman et al. J. Clin. Invest. 107: 1117-1126, 2001), syncytiotrophoblasts (Udagawa et al. Placenta 19:217-223, 1998), synoviocytes (Sugiyama et al. FEBS Lett. 517: 121-128, 2002), and endothelial cells (Iino et al. Arterioscler. Thromb. Vasc. Biol. 18: 40-46, 1998) synthesize and secrete TFPI-2, primarily into their extracellular matrix. Three variants/isoforms of molecular mass 32 kDa, 30 kDa and 27 kDa are synthesized by these cells and are thought to represent differentially glycosylated forms (Rao et al., Arch. Biochem. Biophys. 335:45-52, 1996).
TFPI-2 exhibits inhibitory activity towards a broad spectrum of proteinases including trypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikrein and the factor VIIa-tissue factor complex. However, TFPI-2 exhibits little, if any, inhibitory activity towards urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and α-thrombin (Petersen et al., Biochemistry 35: 266-272, 1996). TFPI-2 presumably inhibits proteinases through a P1 arginine residue (R24) in its Kunitz-type domain, since an R24Q TFPI-2 mutant exhibited only 5-10% inhibitory activity toward trypsin, plasmin and the factor VIIa-tissue factor complex (Kamei et al. Thromb. Res. 94: 147-152, 1999). Recently, TFPI-2 expression by a stably-transfected human high-grade glioma cell line SNB19 resulted in a diminished capacity to form tumors relative to their parental control or mock-transfected SNB19 cells following intracerebral injection of these cells into mice (Konduri et. al., Oncogene 20:6938-6945, 2001). This latter study provides strong experimental evidence that down-regulation of TFPI-2 by tumor cells, presumably through hypermethylation of the TFPI-2 promoter, plays a significant role in the invasive properties of human gliomas.
Plasmin is known to degrade fibrinogen after surgery. Bovine pancreatic trypsin inhibitor (BPTI), also known commercially as aprotinin or Trasylol® (commercially available from Bayer Corporation, West Haven, Conn.), is widely used in the clinic post-operatively by anesthesiologists for general surgery patients and patients undergoing cardiopulmonary bypass surgery to inhibit the degradation of fibrinogen (fibrinolysis) by plasmin arising through activation of the fibrinolytic pathway. Aprotinin inhibits the activity of plasmin. However, aprotinin, being of bovine origin, precipitates episodes of severe anaphylaxis on some occasions (0.5-1%). Accordingly, there is still a need in the art for improved formulations having antifibrinolytic activity that does not produce the undesirable side effects associated with traditional antifibrinolytic compositions.